Mutagenesis is a powerful research tool whereby genetic information is deliberately changed in a stable manner (see, e.g., Hutchinson et al. (1978) J. Biol. Chem. 253:6551; and Razin et al. (1978) Proc. Natl. Acad. Sci. USA 75:4268). Mutagenesis may be performed experimentally by employing the use of recombinant DNA technology and used to introduce specific mutations in a gene to study the effects on its encoded protein. By comparing the properties of a wild-type protein and the mutants generated, it may be possible to identify individual amino acids or domains of amino acids that may be important for the structural integrity and/or biochemical function of the protein (e.g., binding and/or catalytic activity).
Methods of mutagenesis may be random (e.g., error prone PCR) or deliberate (e.g., site directed mutagenesis). A common feature of many methods of mutagenesis is the use of synthetic primers (e.g., oligonucleotides) carrying desired changes in a nucleotide sequence at the site of mutagenesis. For example, saturation mutagenesis uses primers with one or more degenerate codons (e.g., a NNN, a NNK or a NNS codon) that codes for all possible amino acid substitutions at one or more sites in the parent nucleic sequence. Such degenerate codons may code for 32 to 64 unique codons which collectively may encode 20 amino acids in a redundant fashion (e.g., multiple codons for the same amino acid) and a stop codon. For example, given the degeneracy of the genetic code some amino acid residues may be overrepresented (e.g., Arg, Leu, and Ser). This technique depends on screening a large number of variants and may require multiple large libraries using phage or ribosomal display to explore the variants. Another technique, error prone PCR uses polymerase to introduce mutations at random positions in a parent nucleic acid sequence. This technique may introduce mutations outside of an area of interest (e.g., a binding pocket such as a CDR) and necessitate backmutation to identify a productive mutation. Accordingly, methods for mutagenesis are desired that produce manageable libraries comprising targeted mutations at one or more positions in a parent nucleic acid sequence.